Recombinant adenoviruses, use thereof for preparing AAVS, complementary cell line, and pharmaceutical compositions containing said adenoviruses

ABSTRACT

A recombinant adenovirus in which the expression of a nucleic acid sequence coding for at least one homologous or heterologous gene of viral origin is placed under the control of an inducible promoter, is disclosed. The use of such recombinant adenoviruses for preparing AAVs, and a complementary cell line and preparation method therefor, are also disclosed. Furthermore, pharmaceutical compositions containing such an adenovirus are disclosed.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims the prioritybenefit of U.S. application Ser. No. 08/981,354, now U.S. Pat. No.6,420,170, filed Dec. 19, 1997, which claims the benefit of PCTapplication PCT/FR96/00968, filed Jun. 20, 1996, which claims thebenefit of prior French application FR 95 07570 filed Jun. 23, 1995.

The present invention relates to new viral vectors, to their preparationand to their uses. It also relates to pharmaceutical compositionscontaining the said viral vectors.

Gene therapy consists in correcting a deficiency or an abnormality(mutation, aberrant expressions, and the like) by introducing geneticinformation into the cell or organ affected. This genetic informationmay be introduced either in vitro into a cell extracted from the organ,the modified cell then being reintroduced into the body, or directly invivo into the appropriate tissue. In this second case, differenttechniques exist, including various techniques of transfection involvingcomplexes of DNA and DEAE-dextran (Pagano et al., J. Virol. 1 (1967)891), of DNA and nuclear proteins (Kaneda et al., Science 243 (1989)375) and of DNA and lipids (Felgner et al., PNAS 84 (1987) 7413), theuse of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), andthe like.

More recently, the use of viruses as vectors for gene transfer has beenseen to be a promising alternative to these physical transfectiontechniques. In this connection, different viruses have been tested fortheir capacity to infect certain cell populations. This appliesespecially to retroviruses (RSV, HMS, MMS, and the like), the HSV virus,adeno-associated viruses and adenoviruses.

As regards adenoviruses more especially, the latter are lineardouble-stranded DNA viruses approximately 36 kb in size. Their genomecomprises, in particular, an inverted sequence (ITR) at each end, anencapsidation sequence, early genes and late genes (see FIG. 1). Themain early genes are contained in the E1, E2, E3 and E4 regions. Amongthem, the genes contained in the E1 region (E1a and E1b, in particular)are necessary for viral replication. The E4 and L5 regions, for example,are involved in viral propagation, and the main late genes are containedin the L1 to L5 regions. The Ad5 adenovirus genome has been sequencedcompletely and is available on a database (see, in particular, GenebankM73260). Similarly, portions, or in some cases the whole, of the genomeof adenoviruses of different serotypes (Ad2, Ad7, Ad12, and the like)have also been sequenced. These viral vectors advantageously display afairly broad host range, are capable of infecting quiescent cells, donot integrate in the genome of the infected cell and have not beenhitherto associated with significant pathologies in man. In view oftheir properties, they have already been used for gene transfer in vivo.To this end, different vectors derived from adenoviruses have beenprepared, incorporating different genes (β-gal, OTC, α₁-AT, cytokines,and the like).

Naturally, all of these viral vectors contain numerous viral genes whoseexpression is, on the other hand, not desirable in gene therapy. It isessential to control in vivo the non-expression of wild-type viral genesand/or of proteins which are derived therefrom and which are liable toinduce an immune and/or inflammatory response which is undesirable oreven thoroughly deleterious with respect to the body being treated.

For these purposes, the viral vector constructions currently proposedare modified so as to render the said vectors incapable of replicatingautonomously in the target cell. They are said to be defective.Generally, the genome of defective viruses hence lacks at least thesequences necessary for replication of the said virus in the infectedcell. These regions may be either removed (wholly or partially), orrendered non-functional, or replaced by other sequences, and inparticular by a sequence coding for a molecule of therapeutic interest.Preferably, the defective virus nevertheless retains the sequences ofits genome which are necessary for encapsidation of the viral particles.

In the particular case of recombinant adenoviruses, the constructionsdescribed in the prior art are generally adenoviruses from which the E1(E1a and/or E1b) and possibly E3 regions have been deleted, in whichregions the heterologous DNA sequences are inserted (Levrero et al.,Gene 101 (1991) 195; Gosh-Choudhury et al., Gene 50 (1986) 161). Otherconstructions contain a deletion in the E1 region and of a non-essentialportion of the E4 region (WO 94/12649). These defective recombinantadenoviruses may be prepared in different ways, employing or otherwise acompetent cell line capable of complementing all the defective functionsessential for replication of the recombinant adenovirus. At the presenttime, the vectors derived from adenoviruses are generally produced in acomplementation line (line 293) in which a portion of the adenovirusgenome has been integrated. More specifically, line 293 contains theleft-hand end (approximately 11–12%) of the adenovirus serotype 5 (Ad5)genome, comprising the left-hand ITR, the encapsidation region and theE1 region, including E1a, E1b and a portion of the region coding for thepIX protein. This line is capable of trans-complementing recombinantadenoviruses which are defective for the E1 region, that is to saylacking all or part of the E1 region, necessary for replication.

However, during the production of these defective viral vectors, it isnot possible to rule out completely the possibility of recombinationsgenerating replicative viral particles, or in vivotrans-complementations by E1 type cellular functions. It is obvious thatthis type of event is completely incompatible with their subsequent usein gene therapy. The presence in vivo of replicative viral particles mayhave highly deleterious consequences, such as, for example, theinduction of a viral propagation and production of an uncontrolleddissemination with risks of inflammatory reaction, recombination, andthe like.

Concomitantly, it is essential to prevent in vivo the expression ofcorresponding viral proteins. Although the latter do not necessarilydisplay a toxic character with respect to the cell, they are also highlyundesirable since they are also liable to induce immune system responsesof the inflammation type and/or fevers which are detrimental to the bodybeing treated (D. Y. Schwarz, (1995), P.N.A.S. 92, 1401–1405; J. F.Engelhardt, (1994), Human Gene Therapy, 5, 1217–1229 and (1994) P.N.A.S.91, 6196–6200; Y. Yang, (1994), Immunity, 1, 433–442, (1995) J; Virol.,69, 2004–2015 and Nature Genetics, (1994) 7, 362–369).

The objective of the present invention is specifically to provide anapproach enabling these drawbacks to be remedied, and the inventionproves most especially useful for preparing batches of adenovirus typeviruses displaying enhanced safety since, in particular, they lackreplicative viral particles.

Unexpectedly, the Applicant demonstrated that it was possible, using anovel promoter system, to control effectively the expression of viralgene, which expression is effective in vitro during viral productionbut, on the other hand, subsequently ineffective in vivo when the saidrecombinant viruses are used therapeutically.

More specifically, the present invention relates to a recombinantadenovirus in which the expression of at least one homologous orheterologous gene of viral origin is controlled by an induciblepromoter.

For the purposes of the present invention, inducible promoter isunderstood to mean any promoter whose activity is initiated by thepresence of an external chemical and/or biological agent, which agent,in the context of the present invention, displays, in addition, low oreven zero toxicity. “External” is understood to mean that the chemicaland/or biological agent does not naturally exist in the cells treatedwith the claimed adenovirus.

As inducible promoters capable of being employed according to thepresent invention, traditional promoters such as those responding toheavy metals (CRC Boca Raton, Fla. (1991), 167–220; Brinster et al.Nature (1982), 296, 39–42), to thermal shocks, to hormones (Lee et al.P.N.A.S. USA (1988), 85, 1204–1208; (1981), 294, 228–232; Klock et al.Nature (1987), 329, 734–736; Israël and Kaufman, Nucleic Acids Res.(1989), 17, 2589–2604) or to chemical agents of the glucose, lactose,galactose or antibiotic type may be mentioned in particular.

Very recently, a tetracycline-inducible promoter which is especiallyadvantageous in the context of the present invention has been described.

This promoter, termed tetracycline-inducible promoter, comprises aminimal promoter linked operationally to one or more tetracyclineoperator(s). The binding of a so-called “transcription activator”protein to the tetracycline operator sequences, which binding isestablished only in the presence of tetracycline or one of itsanalogues, is the event which permits the activation of the minimalpromoter and hence the transcription of the associated viral gene orgenes.

As regards, more especially, the so-called transcription activatorprotein, this is hence characterized by its ability to bind, in thepresence of tetracycline, to the operator sequences of thetetracycline-inducible promoter, and its capacity to activate theminimal promoter. More preferably, the protein in question consists oftwo polypeptides, a first polypeptide which binds to the tet operatorsequences in the presence of tetracycline or an analogue of the latter,and a second polypeptide whose function is more specifically to activatethe said transcription. The first polypeptide of the so-calledtranscription activator protein is a tetracycline repressor mutated soas to manifest a behaviour opposite to that of a wild-type repressor,that is to say it binds to the tet operator sequences only in thepresence and not in the absence of tetracycline. As regards the secondpolypeptide, this is preferably the activation domain of herpes simplexvirus protein 16.

In the case where the inducible promoter used is, for example, induciblewith glucose or galactose, it is possible to envisage employing atranscription activator constructed on this model, that is to say, forexample, Glu-VP16 or Gal4-VP16.

According to a preferred embodiment of the invention, the induciblepromoter employed is a promoter which is inducible with tetracycline orone of its analogues, as described above.

For the purposes of the present invention, a tetracycline-induciblepromoter comprises a minimal promoter linked operationally to aso-called regulatory sequence comprising at least one operator fortetracycline, “tet operator”, or for one of its analogues.

Tetracycline analogue is understood to cover any compound displayingstructural homologies with tetracycline and which is capable of bindingto its receptor bound to the trans-activation domain of the so-calledtranscription activator protein presented above, with a Ka of at leastapproximately 10⁶ M⁻¹. As analogues capable of being used according tothe present invention, doxycycline, chlorotetracycline andanhydrotetracycline may be mentioned in particular.

Minimal promoter is understood to denote any promoter sequence which, onits own, is not capable of effectively procuring the transcription ofthe DNA sequence which is associated with it. The activity of such apromoter proves to be completely dependent on the binding of thetranscription activator protein to the so-called regulatory sequence inthe presence of tetracycline. In fact, this minimal promoter has aboveall the function of orienting the transcription. From this standpoint,it is preferably located upstream of the viral sequence so as to form acontinuous nucleotide sequence with the latter.

This minimal promoter may be derived from the human cytomegalovirusimmediate-early promoter, and more preferably lies between nucleotides+75 and −53 or +75 and −31. However, it is also possible to employ,according to the invention, a minimal promoter derived from aconventional promoter such as, for example, the one that activates thetranscription of the gene coding for thymidine kinase.

A conventional promoter may also be rendered minimal by means of one ormore genetic mutations which render it incapable of effectivelyprocuring on its own the transcription of the gene which is associatedwith it. A minimal promoter derived directly from the promoter naturallyresponsible for the expression of the viral gene in question may also beemployed in the context of the present invention. It is also possible toenvisage the use of a so-called “TATA-less” promoter as described by E.MARTINEZ et al. (EMBO Journal, (1994), 13, No. 13, 3115–3126), so as toobtain the lowest possible background baseline in the uninducedsituation.

Generally speaking, this minimal promoter is placed upstream of thenucleotide sequence whose expression it controls, as a replacement orotherwise for its natural promoter. The promoter belonging to thenucleic acid sequence can, in effect, remain present, but in a formwhich is inactivated or rendered non-functional by different techniquesknown to a person skilled in the art, and in particular by elimination,deletion and/or addition of one or more bases.

According to a particular embodiment of the invention, the minimalpromoter is derived from the thymidine kinase minimal promoter of herpessimplex virus (McKnight et al. (1984) Cell 37:253–262). It is thendesignated Tk.

More preferably, it is represented wholly or partially by one of thesequences shown as SEQ ID No. 1 or No. 2 or one of their derivatives.

For the purposes of the present invention, the term derivative denotesany sequence obtained by modification of a genetic and/or chemicalnature of given sequences and which retains the desired activity.Modification of a genetic and/or chemical nature should be understood tomean any mutation, substitution, deletion, addition and/or modificationof one or more nucleic acid.

As regards the so-called regulatory sequence, this comprises at leastone operator for tetracycline or one of its analogues. The operator oroperators are recognized by the transcription activator in the presenceof tetracycline and hence, as a result, permit the activation of theminimal promoter.

The tet operator sequences which can be employed may be chosen, inparticular, from those described by Hillen and Wissemann(Protein-Nucleic Acid Interaction, Saeger and Heinemann, eds.,Macmillan, London, (1989) 10, 143–162), Waters et al. (Nucleic AcidsRes. (1983), 11, 525–539), Stüber et al. (P.N.A.S. USA, (1981), 78,167–171), Unger et al. (Nucleic Acids Res. (1984), 12, 7693–7703) andTovar et al. (Mol. Gen. Genet. (1988), 215, 76–80).

The regulatory sequence may comprise a single tet operator sequence or,on the contrary, several tet operator sequences, which can number asmany as 10 depending on whether or not it is desired to increase theregulation of transcription. According to a particular embodiment of theinvention, the regulatory sequence employs 2 tet operator sequences. Itwill then be termed Op2.

More preferably, the regulatory sequence is represented wholly orpartially by one of the sequences shown as SEQ ID No. 3 or No. 4 or oneof their derivatives.

Traditionally, this regulatory sequence is linked operationallyupstream, that is to say at the 5′ end of the minimal promoter, so as topermit the transcription of the gene of viral origin in the presence ofthe complex formed by the transcription activator and its tetracyclineligand. The structure thus comprises, successively, in the 5′ to 3′orientation, the regulatory sequence, bound directly or otherwise to theminimal promoter, the minimal promoter and the gene of viral origin.However, it is also possible to envisage placing this regulatorysequence, within the minimal promoter, downstream of the viralnucleotide sequence to be transcribed, that is to say at its 3′ end. Theorder of succession is then, in the 5′ to 3′ direction, minimalpromoter, viral gene and regulatory sequence.

According to a preferred embodiment of the invention, thetetracycline-inducible promoter links a regulatory sequence representedby Op2 to the thymidine kinase minimal promoter termed Tk. It is in thisparticular case identified below under the name Op2/Tk. More preferably,the inducible promoter employed according to the invention isrepresented wholly or partially by SEQ ID No. 5 or one of itsderivatives.

This tetracycline-inducible promoter Op2/Tk, and more especially the onerepresented wholly or partially by SEQ ID No. 5 or one of itsderivatives, also constitute one of the subjects of the presentinvention.

Consequently, the expression of the viral gene or genes linkedoperationally, in the claimed adenovirus, to an inducible promoter iscompletely dependent on the binding of the complex formed by thetranscription activator and tetracycline to the regulatory sequence ofthe said promoter.

This binding is effective only in the presence of tetracycline. In theabsence of tetracycline or of any analogue of the latter, no binding isestablished between the regulatory sequence and the transcriptionactivator. No transcription of the viral sequence bound to the minimalpromoter ensues. What is more, advantageously, the agent inducingtranscription does not have to be present continuously.

One of the subjects of the present invention relates more especially toan adenovirus comprising at least one homologous, that is to sayadenoviral, gene whose expression is controlled by an induciblepromoter, and more preferably by a tetracycline-inducible promoter.

Thus, in a particular embodiment, the subject of the present inventionis a recombinant adenovirus in which at least one genomic regionessential for viral replication and/or propagation is placed wholly orpartially under the control of a tetracycline-inducible promoter. Theregion essential for viral replication and/or propagation according tothe present invention is advantageously chosen from all or part of theE4, E2 region, the IVa2 region and/or the L5 region, and the like.

According to an especially advantageous embodiment, the recombinantadenoviruses of the present invention comprise all or a functionalportion of the E2 or E4 regions as sequences necessary for replicationand/or propagation. More especially, as regards the E4 region, theimportant genes are the ORF3, ORF6 and ORF6/7 genes.

The E2 region is involved in the regulation of the viral DNA. This E2region consists of two transcription subunits E2A and E2B.

The E4 region is involved in regulation of the expression of the lategenes, in the stability of the late nuclear RNAs, in abolishing theexpression of the host cell's proteins and in the efficacy of thereplication of the viral DNA. Mutants lacking E4 are incapable ofpropagating. E4 thus constitutes a region essential for viralpropagation. This E4 region consists of 7 open reading frames,designated ORF1, ORF2, ORF3, ORF4, ORF3/4, ORF6 and ORF6/7 (FIG. 2).Among these, ORF3 and ORF6 are the two genes essential for viralpropagation. Each of these genes is capable of inducing viralpropagation, ORF6, however, playing a larger part therein than ORF3(Huang and Hearing (1988), J. Virol. 63, 2605).

In a particular embodiment, in the vectors of the invention, the wholeof the region in question is placed under the control of atetracycline-inducible promoter. In the particular case of the E2region, the region in question can be a fragment corresponding to the72K cDNA, to the 140K polymerase cDNA or to the 87K pre-terminal proteincDNA. As regards the E4 region, the region in question can be, inparticular, the Taq1-Bgl2 fragment corresponding to nucleotides35576-32490.

In another particular embodiment, only the expression of a functionalportion of these regions, that is to say sufficient to permit viralpropagation, is controlled. In the particular case of E4, this portioncomprises at least one functional ORF3 or ORF6 gene. Preferably, thefunctional portion of E4 consists essentially of ORF6. As an example,the Bgl2 fragment, lying between positions 34115 and 32490 andcontaining the sequences of the ORF6 and ORF7 of Ad5, may be positioneddownstream of an inducible promoter as defined according to theinvention.

In another particular embodiment of the present invention, the essentialregion consists of the region coding for the IVa2 protein, and forexample its cDNA. In another embodiment, the region coding for the IVa2protein is included in a BglII-NruI fragment corresponding tonucleotides 3328 to 6316 on the wild-type Ad5 adenovirus sequence, aDraI-NlaIII fragment corresponding to nucleotides 4029 to 5719 or a DraIto XhoI fragment corresponding to nucleotides 4029 to 5788.

According to a preferred embodiment of the invention, the promoters ofthe regions essential for viral propagation are replaced within theviral genome by an inducible promoter, and more preferably by atetracycline-inducible promoter.

In a first particular embodiment, the recombinant adenoviruses of theinvention carry a deletion of all or part of the E1 gene and possess theE4 region, wholly or partially, under the control of atetracycline-inducible promoter, preferably of the Op2/Tk type.

In another particular embodiment, the recombinant adenoviruses of theinvention carry a deletion of all or part of the E1 gene and possess theE2 region wholly or partially under the control of atetracycline-inducible promoter, preferably of the Op2/Tk type.

Still according to a preferred embodiment, the recombinant adenovirusesof the invention carry a deletion of all or part of the E1 and E2 genesand possess the E4 region wholly or partially under the control of atetracycline-inducible promoter, preferably of the Op2/Tk type.

In an especially advantageous variant, the recombinant adenoviruses ofthe invention carry a deletion of all or part of the E1 and E4 genes andpossess the E2 region wholly or partially under the control of atetracycline-inducible promoter, preferably of the Op2/Tk type.

Advantageously, the recombinant adenoviruses of the invention contain,in addition, a heterologous nucleic acid sequence containing one or moretherapeutic genes whose transfer to a cell, organ or body and/orexpression therein is sought.

Therapeutic genes which may be transferred in this way are any genewhose transcription and, where appropriate, translation in the targetcell generate products having a therapeutic effect.

Such genes can be, in particular, ones coding for proteinaceous productshaving a therapeutic effect. The proteinaceous product thus encoded canbe a protein, a peptide, and the like. This proteinaceous product can behomologous with respect to the target cell (that is to say a productwhich is normally expressed in the target cell when the latter does notdisplay any pathology). In this case, the expression of a protein makesit possible, for example, to compensate for an insufficient expressionin the cell or for the expression of a protein that is inactive orpoorly active as a result of a modification, or alternatively tooverexpress the said protein. The therapeutic gene can also code for amutant of a cellular protein, having enhanced stability, modifiedactivity, and the like. The proteinaceous product can also beheterologous with respect to the target cell. In this case, an expressedprotein can, for example, supplement or supply an activity which isdeficient in the cell, enabling it to combat a pathology.

Among products which are therapeutic for the purposes of the presentinvention, there may be mentioned, more especially, enzymes, bloodderivatives, hormones, lymphokines, namely interleukins, interferons,TNF, and the like (FR 92/03120), growth factors, neurotransmitters ortheir precursors or synthetic enzymes, trophic factors, namely BDNF,CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, and the like;apolipoproteins, namely ApoAI, ApoAIV, ApoE, and the like (FR 93/05125),dystrophin or a minidystrophin (FR 91/11947), tumour-suppressing genes,namely p53, Rb, Rap1A, DCC, k-rev, and the like (FR 93/04745), genescoding for factors involved in coagulation, namely factors VII, VIII,IX, and the like, suicide genes, namely thymidine kinase; cytosinedeaminase, and the like; or alternatively all or part of a natural orartificial immunoglobulin (Fab, ScFv, and the like), and the like.

The therapeutic gene can also be an antisense gene or sequence whoseexpression in the target cell enables the expression of cellular genesor the transcription of cellular mRNA to be controlled, for instanceribozymes. Such sequences can, for example, be transcribed in the targetcell into RNAs complementary to cellular mRNAs, and can thus block theirtranslation into protein, according to the technique described in PatentEP 140,308.

The therapeutic gene can also be a gene coding for an antigenic peptidecapable of generating an immune response in man. In this particularembodiment, the invention hence makes it possible to produce vaccinesenabling humans to be immunized, in particular against microorganisms orviruses. Such antigenic peptides can be, in particular, specific to theEpstein-Barr virus, the HIV virus, the hepatitis B virus (EP 185,573) orthe pseudorabies virus, or alternatively tumour-specific (EP 259,212).

Generally, the heterologous nucleic acid sequence also comprises atranscription promoter region which is functional in the infected cell,as well a region located at the 3′ end of the gene of interest and whichspecifies a transcription termination signal and a polyadenylation site.These elements collectively constitute the expression cassette. Asregards the promoter region, this can be a promoter region naturallyresponsible for the expression of the gene in question when the regionis capable of functioning in the infected cell. Regions of differentorigin (responsible for the expression of other proteins, or evensynthetic) are a further possibility. In particular, such regions can bepromoter sequences of eukaryotic or viral genes. For example, they canbe promoter sequences originating from the genome of the cell which itis desired to infect. Similarly, they can be promoter sequencesoriginating from the genome of a virus, including the adenovirus used.In this connection, the promoters of E1A, MLP, CMV, RSV, and the like,genes may be mentioned as examples. In addition, these promoter regionsmay be modified by the addition of activator or regulatory sequences orsequences permitting a tissue-specific or -preponderant expression.Moreover, when the heterologous nucleic acid does not contain promotersequences, it may be inserted into the genome of the defective virusdownstream of such a sequence.

Moreover, the heterologous nucleic acid sequence can also contain,especially upstream of the therapeutic gene, a signal sequence directingthe therapeutic product synthesized into the pathways of secretion ofthe target cell. This signal sequence can be the natural signal sequenceof the therapeutic product, but it can also be any other functionalsignal sequence, or an artificial signal sequence.

This nucleic acid sequence is preferably present in the E1, E3 or E4regions, in addition or as a replacement for deleted sequences.

A second main subject of the present invention is an adenoviruscontaining at least one heterologous gene of viral origin whoseexpression is controlled by an inducible promoter, and more preferably atetracycline-inducible promoter.

According to a preferred embodiment of the invention, the heterologousgene of viral origin is or is derived from a gene of the genome of anAAV or one of its functional homologues.

AAVs are relatively small-sized DNA viruses which integrate in thegenome of the cells they infect, stably and site-specifically. They arealso capable of infecting a broad range of cells without inducing aneffect on cell growth, morphology or differentiation. Moreover, theyappear not to be involved in pathologies in man. The AAV genome has beencloned, sequenced and characterized. It comprises 4,680 bases, andcontains an inverted repeat region (ITR) of approximately 145 bases ateach end, serving as origin of replication for the virus. The remainderof the genome is divided into two essential regions carrying theencapsidation functions: the left-hand portion of the genome, whichcontains the rep gene involved in viral replication and the expressionof the viral genes; the right-hand portion of the genome, which containsthe cap gene coding for the capsid proteins of the virus. Threepromoters have been localized therein and named according to theirapproximate position in map units p5, p19 and p40. Four proteins are atleast synthesized from the rep region and have been named on the basisof their apparent molecular mass Rep78, Rep68, Rep52 and Rep40. The 2mRNAs transcribed from the p5 promoter are used for the synthesis ofRep78 and Rep68. Rep52 and Rep40, for their part, are synthesized frommessengers originating from the p19 promoter. As regards the cap genemore especially, this codes for the envelope proteins of the virus (VP1,VP2 and VP3). VP3 is the preponderant capsid protein, and its amino acidsequence is contained in those of two larger but less abundant proteinsVP1 and VP2 (make a diagram). The rep and cap genes have beencharacterized and their respective sequences described in the literature(Srivastava et al., J. Virol. 45 (1983) 555).

The use of vectors derived from AAVs for gene transfer in vitro and invivo has been described in the literature (see, in particular, WO91/18088; WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488,528).Generally, the constructions used in gene therapy contain a deletion ofthe rep and/or cap genes which are replaced by a gene of interest.

In order to replicate, AAVs require the presence of a helper viruscapable of trans-complementing the functions necessary for theirreplication. This can be, in particular, an adenovirus, a herpesvirus ora vaccinia virus. (In the absence of such a helper virus, AAVs remain inlatent form in the genome of infected cells, but cannot replicate andthus cannot produce viral particles.) Traditionally, recombinant AAVsare hence produced by cotransfection, into a cell line infected with ahuman helper virus (for example an adenovirus), of a plasmid containingthe gene of interest flanked by two AAV inverted repeat regions (ITR)and a plasmid carrying the AAV encapsidation genes (rep and cap genes).Coinfection with the adenovirus initiates a cascade of events which endin the production of high titres of AAV and substantially decrease theproduction of adenovirus. This cascade starts with the synthesis of theproduct of the E1a gene, which induces transcription from the p5 and p19promoters and leads to the synthesis of a small amount of Rep proteins.One or more Rep proteins synthesized from p5 then induce the synthesisof mRNA in more abundant amounts from the 3 promoters at a much greaterlevel and in a coordinated manner. In the absence of adenovirus, the AAVgenome is either lost or integrated in the host's chromosome. Genesother than E1A of the adenovirus are also necessary for an effectiveexpression of the AAV genes.

Advantageously, the Applicant demonstrated that it was possible to placeeffectively at least the expression of one of the viral genes of the AAVunder the control of an inducible promoter in an adenovirus, and morepreferably to control the expression of the AAV encapsidation functions,especially the expression of the rep and/or cap genes, or of anyfunctional homologous gene.

A functional homologue corresponds to any gene obtained by modification(mutation, elimination, addition, and the like) of the rep or cap genesand displaying an activity of the same nature. Such functionalhomologous genes can also be genes obtained by hybridization fromnucleic acid libraries by means of probes corresponding to the rep orcap genes. As a mutated rep gene capable of being controlled accordingto the invention, its mutant in1177 described in the publication Y. Yanget al. ((1992) Journal of virology, 6058–6069), and derived from aninsertion of serines between codons 286 and 287, may be mentioned moreespecially.

According to a preferred embodiment of the invention, the induciblepromoter employed is a tetracycline-inducible promoter as defined above.

Such an adenovirus is advantageous in several ways: from the standpointof manipulations, it considerably simplifies the method for preparingstocks of AAV. In effect, in this particular case, essentially only thesaid adenovirus containing the rep and cap genes under the control ofthe inducible promoter, a recombinant AAV and an appropriate cell lineare employed. Lastly, the expected titres of AAV from such an adenovirusprove greater than those obtained according to a conventional method.

The inducible promoter can, in particular, be introduced as areplacement for one of the promoters normally leading to the expressionof the gene or genes in question, and especially as a replacement forthe p5, p19 or p40 promoter. Since the p5 promoter appears to be the onemost involved in the initiation of the cascade of events leading to theproduction of the virus, its replacement by a tetracycline-induciblepromoter, preferably of the Op2/Tk type, is more preferably undertaken.Advantageously, such a construction enables the expression of rep andcap to be blocked in the absence of tetracycline.

The AAV encapsidation functions under the control of an induciblepromoter may be introduced into different regions of the genome of theclaimed adenovirus. Advantageously, the encapsidation functions areinserted into a region which does not interfere with the capacity of thevirus to trans-complement AAVs. It is also possible to insert theencapsidation functions into a functional region of the genome of thesaid adenovirus, this region then being supplied in trans, either by aplasmid or by the cell line used. It is possible, for example, to insertthe rep gene, the cap gene or the rep and cap genes in the E1 or E3regions as a replacement for or in addition to the deleted sequences.

In order to abolish any transcriptional leakage due to the proximity ofthe ITR-psi region, a so-called negative regulatory sequence may, inaddition, be introduced. Such a sequence inserted, in particular,between the left-hand ITR and the psi sequence of the claimed adenoviruson the one hand, and the sequence coding for the tetracycline-induciblepromoter, makes it possible to curb any spurious transcriptionalactivation of rep and cap induced, where appropriate, by the enhancerlocated in the left-hand ITR of the adenovirus and the psi sequence. Asnegative sequences which may be employed according to the invention,those identified in the vimentin promoter (Salvetti et al. (1993), Mol.Cell. Biol. 1676–1685), in the interferon promoter (Whitemore et al.(1990), P.N.A.S., 87, 7799–7803), in the cardiac myosin light chain 2gene (Ruoquian-Shen et al. (1991), Mol. Cell. Biol., 1676–1685) and inthe mouse albumin promoter (Herbst et al. (1990), Mol. Cell. Biol.,3896–3905) may be mentioned in particular.

According to a preferred embodiment, the invention relates to arecombinant adenovirus containing an Op2/Tk-rep-cap expression cassette.

The subject of the present invention is also the use of theseadenoviruses integrating a viral sequence of AAV origin under thecontrol of a tetracycline-inducible promoter for preparing AAVs.

In a preferred embodiment, the adenoviruses which are the subjects ofthe invention comprise the ITR sequence and a sequence permittingencapsidation. Preferably, these adenoviruses possess, in addition, anon-functional E1 region.

The inverted repeat sequences (ITR) constitute the origin of replicationof the adenoviruses. They are localized at the 3′ and 5′ ends of theviral genome (see FIG. 1), from where they may be isolated readilyaccording to the traditional techniques of molecular biology known to aperson skilled in the art. The nucleotide sequence of the ITR sequencesof human adenoviruses (especially of the serotypes Ad2 and Ad5) isdescribed in the literature, as are those of canine adenoviruses (inparticular CAV1 and CAV2). As regards the Ad5 adenovirus for example,the left-hand ITR sequence corresponds to the region comprisingnucleotides 1 to 103 of the genome.

The encapsidation sequence (also designated psi sequence) is necessaryfor encapsidation of the viral DNA. This region must hence be present inorder to permit the preparation of defective recombinant adenovirusesaccording to the invention. The encapsidation sequence is localized inthe genome of the adenoviruses, between the left-hand (5′) ITR and theE1 gene (see FIG. 1). It may be isolated or synthesized artificially bytraditional techniques of molecular biology. The nucleotide sequence ofthe encapsidation sequence of human adenoviruses (especially of theserotypes Ad2 and Ad5) is described in the literature, as are those ofcanine adenoviruses (in particular CAV1 and CAV2). As regards the Ad5adenovirus for example, the encapsidation sequence corresponds to theregion comprising nucleotides 194 to 358 of the genome.

According to an especially advantageous embodiment, in the recombinantadenoviruses of the present invention, the E1 region is inactivated bydeletion of a PvuII-BglII fragment extending from nucleotide 454 tonucleotide 3328 on the Ad5 adenovirus sequence. This sequence isavailable in the literature and also on a database (see, in particular,Genebank No. M73260). In another preferred embodiment, the E1 region isinactivated by deletion of a HinfII-Sau3A fragment extending fromnucleotide 382 to nucleotide 3446.

The adenoviruses of the invention may be prepared from adenoviruses ofdiverse origins. There are, in effect, different serotypes ofadenovirus, the structure and properties of which vary somewhat butwhich display a comparable genetic organization. Thus, the teachingsdescribed in the present application may be readily reproduced by aperson skilled in the art for any type of adenovirus.

More especially, the adenoviruses of the invention may be of human,animal or mixed (human and animal) origin.

As regards adenoviruses of human origin, it is preferable to use thoseclassified in group C. More preferably, among the different serotypes ofhuman adenovirus, it is preferable to use adenoviruses type 2 or 5 (Ad2or Ad5) in the context of the present invention.

As mentioned above, the adenoviruses of the invention may also be ofanimal origin, or may contain sequences originating from adenoviruses ofanimal origin. The Applicant has, in effect, shown that adenoviruses ofanimal origin are capable of infecting human cells with great efficacy,and that they are incapable of propagating in the human cells in whichthey have been tested (see Application WO 94/26914). The Applicant hasalso shown that adenoviruses of animal origin are in no waytrans-complemented by adenoviruses of human origin, thereby eliminatingany risk of recombination and propagation in vivo in the presence of ahuman adenovirus, which can lead to the formation of an infectiousparticle. The use of adenoviruses or of regions of adenoviruses ofanimal origin is hence especially advantageous, since the risks inherentin the use of viruses as vectors in gene therapy are even lower.

The adenoviruses of animal origin which may be used in the context ofthe present invention can be of canine, bovine, murine (for example:Mav1, Beard et al., Virology 75 (1990) 81), ovine, porcine, avian oralternatively simian (for example: SAV) origin. More especially, amongavian adenoviruses, there may be mentioned the serotypes 1 to 10 whichare available in the ATCC, such as, for example, the strains Phelps(ATCC VR-432), Fontes (ATCC VR-280), P7-A (ATCC VR-827), IBH-2A (ATCCVR-828), J2-A (ATCC VR-829), T8-A (ATCC VR-830), K-11 (ATCC VR-921) oralternatively the strains referenced ATCC VR-831 to 835. Among bovineadenoviruses, the different known serotypes may be used, and inparticular those available in the ATCC (types 1 to 8) under thereferences ATCC VR-313, 314, 639–642, 768 and 769. There may also bementioned the murine adenoviruses FL (ATCC VR-550) and E20308 (ATCCVR-528), ovine adenovirus type 5 (ATCC VR-1343) or type 6 (ATCCVR-1340), porcine adenovirus 5359, or simian adenoviruses such as, inparticular, the adenoviruses referenced in the ATCC under the numbersVR-591–594, 941–943, 195–203, and the like.

Among the different adenoviruses of animal origin, it is preferable inthe context of the invention to use adenoviruses or regions ofadenoviruses of canine origin, and in particular all strains of CAV2adenoviruses [strain Manhattan or A26/61 (ATCC VR-800) for example].Canine adenoviruses have been subjected to many structural studies.Thus, complete restriction maps of CAV1 and CAV2 adenoviruses have beendescribed in the prior art (Spibey et al., J. Gen. Virol. 70 (1989)165), and the E1a and E3 genes as well as the ITR sequences have beencloned and sequenced (see, in particular, Spibey et al., Virus Res. 14(1989) 241; Linné, Virus Res. 23 (1992) 119, WO 91/11525).

The present invention relates, in addition, to a method which is usefulfor the preparation of AAV.

More specifically, its subject is a method for preparing AAV,characterized in that it comprises the cotransfection, in the presenceof tetracycline or one of its analogues, of a cell line comprising inits genome the cassette for the expression of a transcription activator,with an adenovirus comprising at least one gene of AAV origin under thecontrol of a tetracycline-inducible promoter, and either a recombinantvirus derived from the AAV or a plasmid carrying a transgene between theITRs of the AAV. The adenovirus is preferably one comprising the rep andcap genes as heterologous viral genes.

The method according to the invention turns to good account the abilityto induce the expression of these rep and cap genes placed under thecontrol of a tetracycline-inducible promoter within an adenovirus, inthe presence of a sufficient amount of tetracycline and a transcriptionactivator.

As explained earlier, this method has the advantage of being simplifiedfrom the standpoint of manipulations compared to a traditional method.In the present case, all that is carried out is a coinfection of a cellline with an adenovirus such as is claimed and a recombinant virusderived from an AAV.

Besides the transformed adenovirus according to the invention, thismethod employs a cell line containing in its genome a cassette for theexpression of the so-called transcription activator protein consistingof a first polypeptide capable of binding, in the presence oftetracycline or one of its analogues, to the regulatory sequence of theinducible promoter present in the adenovirus, combined with a secondpolypeptide which activates transcription.

As regards, more especially, the so-called transcription activatorprotein, this is hence characterized by its ability to bind, in thepresence of tetracycline, to the so-called regulatory sequence and itscapacity to activate the minimal promoter which is associated with it.As explained above, it is a protein consisting of two polypeptides, afirst polypeptide which binds to the tet operator sequences in thepresence of tetracycline or an analogue of the latter, and a secondpolypeptide whose function is more specifically to activate the saidtranscription.

According to a favoured embodiment of the invention, the firstpolypeptide of the so-called transcription activator protein is atetracycline repressor mutated so as to manifest a behaviour opposite tothat of a wild-type repressor, that is to say it binds to the tetoperator sequences only in the presence and not in the absence oftetracycline. This type of mutation may be performed according totraditional biological techniques of the mutagenesis type. Thedifference in amino acids between the wild-type repressor and themutated repressor according to the present invention can consist of asubstitution, deletion and/or addition of one or more amino acids. Ithas the effect of endowing the repressor thus transformed with twofunctional properties: it can bind to the regulatory sequencerepresented by tetracycline operators by analogy with the wild-typerepressor; in contrast, it is regulated inversely by tetracycline.

Numerous classes of wild-type tetracycline repressors have already beendescribed in the literature, among which classes A, B, C, D and E may bementioned in particular. As a representative example of theserepressors, the repressor termed Tn10 which belongs to class B may bementioned more especially. According to a preferred embodiment of theinvention, the repressor employed is derived from this wild-typerepressor Tn10. More specifically, it is a Tn10 repressor mutated in atleast one amino acid localized at position 71, 95, 101 or 102.

More preferably, it possesses wholly or partially the amino acidsequence shown as SEQ ID No. 8. It will termed TetR.

As regards the second polypeptide present in the so-called transcriptionactivator protein, this can be any already known transcriptionalactivation domain. According to a preferred embodiment of the invention,it is the activation domain of herpes simplex virus protein 16, moreespecially the 130 amino acids of the C-terminal end of VP16, and morepreferably the 11 amino acids of this C-terminal end of VP16 oralternatively peptide portions of the C-terminal portion of VP16(Sceipel K; et al. EMBO J. 1992; 13, 4961–4968) or derivatives.

In the case of the claimed method for producing AAV, the cassette forthe expression of this transcription activator is preferably integratedin the genome of a cell line 293.

According to a preferred embodiment of the invention, the expression ofthis transcription activator is also placed, in the cell line, under thecontrol of a promoter which is inducible with tetracycline or one of itsanalogues as is defined above. More preferably, the cell line inquestion is a cell line 293 integrating in its genome theOp2/Tk-TetR-VP16 cassette.

The subject of the present invention is also a cell line containing inits genome a cassette for the expression of a transcription activator asdefined above, comprising or otherwise an inducible promoter as definedaccording to the invention. More preferably, a cell line is a lineintegrating in its genome the Op2/Tk-TetR-VP16 cassette.

The invention also relates to the use of this type of cell line forproducing adenoviruses according to the invention or AAVs.

The subject of the present invention is also a method for preparingadenoviruses containing at least one of their genes whose expression isunder the control of the tetracycline-inducible promoter.

The defective recombinant adenoviruses according to the invention may beprepared in different ways.

A first method consists in transfecting the DNA of the defectiverecombinant virus prepared in vitro (either by ligation or in plasmidform) into a competent cell line, that is to say one carrying in transall the functions necessary for complementation of the virus, and atranscription activator. These functions are preferably integrated inthe genome of the cell, thereby enabling risks of recombination to beavoided and endowing the cell line with enhanced stability.

Thereafter, the vectors which have multiplied in the presence of asufficient amount of tetracycline or one of its analogues are recovered,purified and amplified according to traditional techniques of molecularbiology.

According to a variant of implementation, it is possible to prepare invitro, either by ligation or in plasmid form, the DNA of the defectiverecombinant virus carrying the appropriate deletions, one or more viralgenes under the control of a tetracycline-inducible promoter and one ormore therapeutic genes. The eliminations are generally carried out onthe DNA of the defective recombinant virus, by performing digestions bymeans of suitable restriction enzymes, followed by ligations, accordingto the techniques of molecular biology, as illustrated in the examples.The viral or therapeutic genes and the inducible promoter may then beinserted into this DNA by enzymatic cleavage followed by ligation, inthe selected regions and in the chosen orientation. The DNA therebyobtained, which hence carries the appropriate deletions, one or moreviral genes under the control of a tetracycline-inducible promoter andone or more therapeutic genes, enables the claimed recombinantadenovirus to be generated directly.

It is also possible to prepare the recombinant virus in successivesteps, permitting the successive introduction of the heterologous genesand the inducible promoter. Thus, the DNA of a first recombinant viruscarrying the appropriate deletions (or a part of the said deletions) andan inducible promoter such as, for example, Op2/Tk is constructed byligation or in plasmid form. This DNA is then used to generate a firstrecombinant virus carrying the said deletions with an induciblepromoter. The DNA of this first virus is then isolated and cotransfectedwith a second plasmid or the DNA of a second defective recombinant viruscarrying the appropriate deletions, in particular a deletion in the E1region, a region permitting homologous recombination and, whereappropriate, a therapeutic gene. This second step thus generates therecombinant virus according to the invention.

The present invention also relates to any pharmaceutical compositioncomprising one or more recombinant adenoviruses as described above. Thepharmaceutical compositions of the invention may be formulated with aview to topical, oral, parenteral, intranasal, intravenous,intramuscular, subcutaneous, intraocular, transdermal, and the like,administration.

Preferably, the pharmaceutical composition contains vehicles which arepharmaceutically acceptable for an injectable formulation. These can be,in particular, sterile, isotonic saline solutions (of monosodium ordisodium phosphate, sodium, potassium, calcium or magnesium chloride,and the like, or mixtures of such salts), or dry, in particularlyophilized, compositions which, on adding sterilized water orphysiological saline, as the case may be, enable injectable solutions tobe formed.

The doses of virus used for the injection may be adapted in accordancewith different parameters, and in particular in accordance with the modeof administration used, the pathology in question, the gene to beexpressed or the desired period of treatment. Generally speaking, therecombinant adenoviruses according to the invention are formulated andadministered in the form of doses of between 10⁴ and 10¹⁴ pfu/ml, andpreferably 10⁶ to 10¹⁰ pfu/ml. The term pfu (plaque forming unit)corresponds to the infectious power of a solution of virus, and isdetermined by infecting a suitable cell culture and measuring, generallyafter 5 days, the number of plaques of infected cells. The techniques ofdetermination of the pfu titre of a viral solution are well documentedin the literature.

The adenoviruses of the invention may be used for the treatment orprevention of numerous pathologies. They are especially advantageous forthe treatment of hyperproliferative pathologies (cancers, restenosis,and the like), by direct injection at the site in question. In thisconnection, the present invention also relates to a method for thedestruction of proliferative cells, comprising the infection of the saidcells or of a portion of them with an adenoviral vector as definedabove. In the case where the suicide gene is a gene conferringsensitivity to a therapeutic agent, the method of destruction accordingto the invention thereafter comprises the treatment of the cells withthe said therapeutic agent. To carry out this method, the subject of theinvention is also the products comprising a recombinant adenovirus asdefined above in which the suicide gene is a gene conferring sensitivityto a therapeutic agent; and the said therapeutic agent as a combinationproduct for use simultaneously, separately or spread over time for thetreatment of hyperproliferative pathologies. More especially, thesuicide gene is a thymidine kinase gene and the therapeutic agent isganciclovir or acyclovir or an analogue.

Recombinant vectors according to the invention possess especiallyattractive properties for use in gene therapy. These vectors combine, ineffect, very superior properties of infection, safety and gene transfercapacity.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be described more completely by means of theexamples and figures which follow, which should be considered to beillustrative and non-limiting.

FIG. 1: Genetic organization of the Ad5 adenovirus. The completesequence of Ad5 is available on a database, and enables a person skilledin the art to select or create any restriction site, and thus to isolateany region of the genome.

FIG. 2: Genetic organization of the E4 region.

FIG. 3: Genetic organization of AAV.

DETAILED DESCRIPTION OF THE INVENTION AND EXAMPLES

General Techniques of Molecular Biology

The methods traditionally used in molecular biology, such as preparativeextractions of plasmid DNA, centrifugation of plasmid DNA in a caesiumchloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol-chloroform extractions of proteins, ethanol or isopropanolprecipitation of DNA in a saline medium, transformation in Escherichiacoli, and the like, are well known to a person skilled in the art andare amply described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F. M. et al. (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, 1987].

Plasmids of the pBR322 and pUC type and phages of the M13 series are ofcommercial origin (Bethesda Research Laboratories).

To carry out ligation, the DNA fragments may be separated according totheir size by agarose or acrylamide gel electrophoresis, extracted withphenol or with a phenol-chloroform mixture, precipitated with ethanoland then incubated in the presence of phage T4 DNA ligase (Biolabs)according to the supplier's recommendations.

The filling in of 5′ protruding ends may be performed with the Klenowfragment of E. coli DNA polymerase I (Biolabs) according to thesupplier's specifications. The destruction of 3′ protruding ends isperformed in the presence of phage T4 DNA polymerase (Biolabs) usedaccording to the manufacturer's recommendations. The destruction of 5′protruding ends is performed by a controlled treatment with S1 nuclease.

In vitro site-directed mutagenesis using synthetic oligodeoxynucleotidesmay be performed according to the method developed by Taylor et al.[Nucleic Acids Res. 13 (1985) 8749–8764] using the kit distributed byAmersham.

The enzymatic amplification of DNA fragments by the so-called PCR[polymerase-catalysed chain reaction, Saiki R. K. et al., Science 230(1985) 1350–1354; Mullis K. B. and Faloona F. A., Meth. Enzym. 155(1987) 335–350] technique may be performed using a “DNA thermal cycler”(Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of the nucleotide sequences may be performed by the methoddeveloped by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977)5463–5467] using the kit distributed by Amersham.

Cell Lines Used

In the examples which follow, the following cell lines were or may beused:

-   -   Human embryonic kidney line 293 (Graham et al., J. Gen. Virol.        36 (1977) 59). This line contains, in particular, integrated in        its genome, the left-hand portion of the Ad5 human adenovirus        genome (12%).    -   KB human cell line: Originating from a human epidermal        carcinoma, this line is available in the ATCC (ref. CCL17),        together with the conditions enabling it to be cultured.    -   Hela human cell line: Originating from a human epithelial        carcinoma, this line is available in the ATCC (ref. CCL2),        together with the conditions enabling it to be cultured.    -   MDCK canine cell line: The conditions of culture of MDCK cells        have been described, in particular, by Macatney et al., Science        44 (1988) 9.    -   gm DBP6 cell line (Brough et al., Virology 190 (1992) 624). This        line consists of Hela cells carrying the adenovirus E2 gene        under the control of the MMTV LTR.

EXAMPLE 1

Construction of an Adenovirus Carrying its E4 Domain Under the Controlof an Op2-Tk Promoter.

1—Construction of the Plasmid pIC20H/Op2-Tk:

This plasmid carries the sequence of the Tk minimal promoter preceded bytwo sequences of the tetracycline operator; these sequences arerecognized by the tetracycline repressor when it is bound totetracycline.

To obtain it, the plasmid pIC20H (Marsh et al., Gene 32 (1984) 481) isdigested with ClaI/BamHI, and the sequence SEQ ID No. 5, comprising twotetracycline operators upstream of a thymidine kinase minimal promoter,is introduced between these two sites.

2—Construction of the Plasmid pIC20H/ITR-Op2Tk

This plasmid is obtained by Cla1 digestion of plasmid pIC20H/Op2Tk andinsertion of an Hpa2 fragment containing the Ad5 ITR (coordinates:1/+122). This fragment comes from the commercial vector pSL1180(Pharmacia) digested with Hind3, into which site the ITR manufactured byPCR is introduced, with Hind3 sites on each side of the amplifiedfragment. There is obtained in the following order: ITR-Op2-TKprom.

3—Construction of the Plasmid pIC20H/ITR-Op2Tk-E4

This plasmid corresponds to plasmid pIC20H/ITR-Op2Tk digested withHind3, into which site the Nhe-Xba1 fragment of PY6 containing the E4region of Ad5 is inserted. The plasmid pPY6, for its part, is obtainedaccording to the following protocol:

A plasmid pPY2 is prepared from plasmid pIC20H. This plasmid pPY2corresponds to the cloning of the Avr2-Sal1 fragment (approximately 1.3kb including the MMTV promoter) of the plasmid pMSG (Pharmacia) betweenthe Xba1 and Sal1 sites of plasmid pIC20H prepared from an E. coli dam+context. The plasmid pPY4 is derived from plasmid pPY2 by deletion of a35-bp fragment after cleavage with BamH1 and Bgl2 followed byreligation. The plasmid pPY5 corresponds to plasmid pIC20H in which theTaq1-Bgl2 fragment including the E4 region of adenovirus type 5, locatedbetween positions 35576 (Taq1) and 32490 (Bgl2), has been cloned betweenthe Cla1 and BamH1 sites. The E4 region of plasmid pPY5 is henceincluded in an EcoRV-Sph1 fragment which is cloned after partialdigestion between the Sma1 and Sph1 sites of plasmid pPY4, therebygenerating the plasmid pPY6.

4—Construction of the Plasmid pIC20H/ITR-Op2Tk-E4-L5

This is obtained by digestion of plasmid pIC20H/ITR-Op2Tk-E4 with Kpn1and Xba1, and insertion of the 3.1-kb Kpn1-Xba1 fragment of Ad5(coordinates: 33595–30470) containing the whole of the L5 region.

5—Construction of the Plasmid pYG4-EP

Plasmid pIC20H/ITR-Op2Tk-E4-L5 is digested with Xba1 and Nru1 to recoverthe corresponding fragment, which carries in order the ITR, Op2, the Tkpromoter, E4 and L5. This fragment is inserted into the Xba1 and Nru1sites of plasmid pYG4, which contains the whole of the sequence of theadenovirus from the Xba1 site to the Sph1 site. This plasmid pYG4-EP isa vector pIC20H into which the Sph1-Xba1 fragment of Ad5 (coordinates:25095–28590) is inserted between its Sph1 and Xba1 sites.

This vector pYG4-EP, from which the E3 adenoviral region has beendeleted, possesses sufficient adenoviral sequences between the Sph1 andXba1 sites to permit complementary recombination of the adenovirus forthe production of a recombinant adenovirus.

6—Construction of the Recombinant Adenovirus

This is carried out by cotransfection of 293/TetR-VP16 cells, preparedaccording to Example 3 below, with plasmid pYG4 linearized by Sph1digestion and with the adenovirus RSV-βgal or an adenovirus carrying atransgene, linearized by Srf1 digestion, in the presence or absence oftetracycline. The selection and amplification of the recombinantadenovirus is then carried out according to traditional virologicaltechniques.

EXAMPLE 2

Construction of the Recombinant Adenovirus Carrying the AAV rep-capGenes under the Control of the Op2/Tk Promoter.

1—Construction of the Intermediate Plasmid pXL2630

This intermediate plasmid enables an EcoRI site to be introduceddownstream of Op2-Tk. The presence of a restriction site at thisposition is of twofold interest. It serves to introduce this promoterupstream of rep-cap after the p5 promoter has been eliminated, and italso enables this hybrid promoter to be inserted upstream of TetR-VP16for the preparation of a transformed cell line 293, as described inExample 3 below.

To this end, plasmid pIC20H/Op2-Tk, obtained according to the protocoldescribed in Example 1, is digested with BamHI, treated with T4 DNApolymerase in order to blunt the ends and then redigested with EcoRV,and the fragment originating from this digestion and carrying the Op2-Tkpromoter is introduced at the EcoRV site of the commercial plasmidpBSSK+. The orientation of the fragment is selected for the presence ofan EcoRI site downstream of the promoter.

3—Introduction of an EcoRI Site at the +1 Position with Respect toTranscription of p5

To eliminate the p5 promoter, an EcoRI site is introduced at the +1position with respect to transcription of the p5 promoter upstream ofthe Rep78 coding sequence by the PCR technique on the plasmid pAV2(Laughlin C., Gene (1983), 23, 69–73). This reaction was carried outusing the oligonucleotides:

-   SEQ ID No. 6 (seq5269): 5′GAATTCTTTTGAAGCGGGAGGTTTGAACGCG 3′ EcoRI-   SEQ ID No. 7 (seq5039): 5′CTCCATGTACCTGGCTGA 3′

The fragment thus generated was introduced into pCRII (Invitrogen) togive the plasmid pMA4. The nucleotide sequence of this fragment wasverified.

4—Construction of the Plasmid pMA6 Carrying the Op2-Tk-rep-cap Junction

This intermediate plasmid enables the joining of the inducible promoterwith rep to be carried out. The SalI-EcoRI fragment of pXL2630 and theEcoRI-NruI fragment of pMA4 are introduced at the XhoI (compatible withSalI) and NruI sites of pIC20R (Marsh et al., Gene 32 (1984) 481) togive plasmid pMA6.

5—Construction of the Plasmid pC01 (FIG. 7 EX94008) Which Contains theLeft-hand Portion of the Ad5 Adenovirus up to the HinfI Site (382), aMultiple Cloning Site and the Sau3A (3446)-NruI (6316) Fragment of theAd5 Adenovirus

5-a/Construction of the Plasmid pCE

The EcoRI-XbaI fragment corresponding to the left-hand end of the Ad5adenovirus genome was first cloned between the EcoRI and XbaI sites ofthe vector pIC19H (Marsh et al., Gene 32 (1984) 481). This generates theplasmid pCA. Plasmid pCA was then cut with HinfI, its 5′ protruding endswere filled in with the Klenow fragment of E. coli DNA polymerase I andit was then cut with EcoRI. The fragment thus generated of plasmid pCA,which contains the left-hand end of the Ad5 adenovirus genome, was thencloned between the EcoRI and SmaI sites of the vector pIC20H (Marsh etal., Gene 32 (1984) 481). This generates the plasmid pCB. Plasmid pCBwas then cut with EcoRI, its 5′ protruding ends were filled in with theKlenow fragment of E. coli DNA polymerase I and it was then cut withBamHI. The fragment thus generated of plasmid pCB, which contains theleft-hand end of the Ad5 adenovirus genome, was then cloned between theNruI and BglII sites of the vector pIC20H. This generates the plasmidpCE, an advantageous feature of which is that it possesses the first 382base pairs of the Ad5 adenovirus followed by a multiple cloning site.

5-b/Construction of the Plasmid pCD′

The Sau3A (3346)-SstI (3645) fragment and the SstI (3645)-NarI (5519)fragment of the Ad5 adenovirus genome were first ligated and clonedbetween the ClaI and BamHI sites of the vector pIC20H, therebygenerating plasmid pPY53. The SalI-TaqI fragment of plasmid pPY53prepared from a dam- context, containing the portion of the Ad5adenovirus genome lying between the Sau3A (3346) and TaqI (5207) sites,was then cloned between the SalI and ClaI sites of the vector pIC20H,thereby generating the plasmid pCA′. The TaqI (5207)-NarI (5519)fragment of the Ad5 adenovirus genome prepared from a dam- context andthe SalI-TaqI fragment of plasmid pCA′ were then ligated and clonedbetween the SalI and NarI sites of the vector pIC20H. This generates theplasmid pCC′. The NarI (5519)-NruI (6316) fragment of the Ad5 adenovirusgenome prepared from a dam- context and the SalI-NarI fragment ofplasmid pCC′ were then ligated and cloned between the SalI and NruIsites of the vector pIC20R. This generates the plasmid pCD′.

5-c/Construction of Plasmid pC01

A partial digestion with XhoI followed by a complete digestion with SalIof plasmid pCD′ generates a restriction fragment which contains the Ad5adenovirus sequence from the Sau3A site (3446) to the NruI site (6316).This fragment was cloned into the SalI site of plasmid pCE. Thisgenerates plasmid pC01.

6—Construction of the Plasmids pMA7 and pMA8

The EcoRV-SnaBI fragment of pMA6, carrying the AAV Op2-Tk-rep-cap-polyA+(up to the SnaBI site, position 4495 on the AAV sequence), is introducedat the EcoRV site of pCO1 in both orientations relative to theadenovirus ITR. The plasmids thereby obtained are designated pMA7(orientation of the cassette in the direction opposite to the adenovirusITR) and pMA8 (same orientation).

7—Construction of the Recombinant Adenovirus Carrying Op2-Tk-rep-cap

This part describes the construction of a defective recombinantadenovirus carrying the AAV Op2-Tk-rep-cap-polyA+ cassette. Thisadenovirus is obtained by cotransfection of plasmid pMA7 or pMA8 with adeficient adenoviral vector, into helper cells (line 293) supplying intrans the functions encoded by the adenovirus E1 (E1A and E1B) regions.

More specifically, the adenoviruses AdMA7 and AdMA8 were prepared byhomologous recombination in vivo between the adenovirus AdRSVβgal andplasmids pMA7 and pMA8 according to the following protocol: plasmid pMA7or pMA8 linearized with NdeI and the adenovirus AdRSVBgal linearizedwith ClaI are cotransfected into line 293 in the presence of calciumphosphate to permit recombination. The recombinant adenoviruses thusgenerated are selected by plaque purification. After isolation, therecombinant adenovirus is amplified in cell line 293, leading to aculture supernatant containing the unpurified defective recombinantadenovirus having a titre of approximately 1010 pfu/ml. For thepurification, the viral particles are centrifuged on a caesium chloridegradient according to known techniques (see, in particular, Graham etal., Virology 52 (1973) 456).

The adenovirus AdMA7 or AdMA8 is stored at −80° C. in 20% glycerol.

8 Construction of the Recombinant Adenovirus Carrying Op2/Tk rep-cappolyA+AAV in the E3 Region:

This part describes the construction of a recombinant adenovirus whichis deleted for E1 and which carries Op2:Tk repcap polyA+AAV in the E3region.

Plasmid pMA28 contains all of the sequence of the Ad(E1-, E3-) carryingOp2:Tk repcap polyA+AAV in the E3 region. It was constructed by means ofrecombination in E. Coli, by, for example, introducing the plasmid pMA24into the strain C2110 (pXL2638) (E1-, E3-), which strain is described inapplication PCT/FR96/00215, which is included herein by reference.

8.1 Construction of the Intermediate Plasmid pMA22:

The Xba1—Xba1 fragment of the plasmid pMA7, carrying Op2:Tk repcappolyA+AAV, was introduced into the Xba1 site of pYG4-EP in place of theE3 region, such that Op2:Tk repcap polyA+AAV is in the inverseorientation to that of the E3 region. The plasmid which is constructedin this way is pMA22.

8.2 Construction of Plasmid pMA24, Which is Used to PerformRecombination in E. Coli:

The Nhe1-Spe1 fragment of pMA22, containing the Op2:Tk repcap polyA+AAVcassette flanked by the adenovirus 27082–28593 and 3471–31509 sequences,was introduced into the compatible site of plasmid pXL2756 in order togenerate plasmid pMA24, which plasmid carries the regions required forrecombination flanking the Op2:Tk repcap polyA+AAV cassette, the B.subtilis sacB gene and the gene for resistance to kanamycin.

8.3 Construction of the Recombinant Adenovirus Carrying Op2:Tk repcappolya+AAV in the E3 Region:

This construction was carried out by means of recombination in E. Coli,by electroporating plasmid pMA24 into strain C2110 (pXL2688) or C2110(pXL2789) and selecting for a second recombination event on LB mediumcontaining sucrose and tetracyclin. A C2110 strain harbouring plasmidpMA28 is thereby obtained.

This plasmid was then transfected, after digestion with Pac1, into 293cells.

EXAMPLE 3

Construction of the Producing Line 293 Op2-Tk-TetR-VP16.

This part describes the construction of a 293 line carrying, integratedin its genome, the cassette of the hybrid trans-activator TetR-VP16under the control of the Op2-Tk promoter. For this purpose, the plasmidpMA2 was constructed in order to establish a line by cotransfection ofthis plasmid pMA2 with a plasmid pMSCV (Hawley et al. J. Exp. Med.(1993), vol. 176, 1149–1163) carrying the neomycin-resistance gene underthe control of the PGK (phosphoglycerate kinase) promoter. pMA2 isconstructed by inserting the SalI-EcoRI fragment of pXL2630 between thecompatible XhoI-EcoRI sites of a plasmid pUHD17.1. Plasmid pUHD17.1 is aplasmid comprising the sequences coding for a mutated tetracyclinerepressor linked operationally to the VP16 sequence. This vector isderived from the vector pUHD15.1 (H. Bujard; P.N.A.S. U.S.A. 1992, 89,55476–5551) which comprises the sequence of the wild-type tetracyclinerepressor combined with the 130 amino acids of the C-terminal end ofherpes simplex virus VP16. A 399-base pair Xba1-Eco471II fragmentcorresponding to amino acids 3 to 135 of the mutated tetracyclinerepressor is exchanged for the corresponding restriction fragment ofpUHD15.1 to yield pUHD17.1.

The line 293 Op2-Tk-TetR-VP16 of the invention was constructed bycotransfection of the chosen cells in the presence of calcium phosphatewith plasmids pMA2 and pMSCV and a construction coding for theglucocorticoid receptor (Hollenberg et al., 1985). More specifically,line 293 cells in dishes 5 cm in diameter were transfected with 1 to 5μg of plasmid pMA2.

Selection of Geneticin-resistant Clones

After transfection of the cells, the latter are washed, the culturemedium (MEM, Sigma) supplemented with foetal calf serum (7% final) isthen added and the cells are incubated for 20 hours. Next day, the cellsare selected in the presence of geneticin G418 (Gibco-BRL, LifeTechnologies) at an effective concentration of 400 mg/l. The geneticinis changed every three days and the selectable clones appear afterapproximately 3 weeks. When all the untransfected cells have died, onlycells which have inserted the resistance gene survive and divide togenerate cell clones. When the cell clones are sufficiently large to bevisible to the naked eye, they are transfer individually to the culturewells of a “24-cavity” culture plate. Each clone is then graduallyamplified in the presence of geneticin, first in the wells of a“12-cavity” culture plate and then of a “6-cavity” culture plate, andthereafter amplified in cell culture dishes. Each cell clone is thenstored by freezing in liquid nitrogen.

A number of clones were isolated, amplified and selected for theircapacity to express a reporter gene, for example lacZ under the controlof the Op2-Tk promoter after adding a suitable concentration oftetracycline. The plasmid used is pMA9, and was constructed byintroducing a StuI-BamHI fragment-of pRSVgalIX carrying the sequencecoding for E. coli β-galactosidase and a nuclear localization signalinto plasmid pMA2 previously linearized with EcoRI; treated withbacteriophase T4 DNA polymerase in order to blunt its ends and thenredigested with BamHI.

Among these clones, those permitting a conditional expression of rep-capcarried by the adenovirus described above and permitting AAV productionat high titres were used as producing line.

1. A recombinant adenovirus wherein a gene of viral origin is placedunder the control of an inducible promoter, wherein the induciblepromoter comprises a minimal promoter linked operationally to atetracycline operator, and the tetracycline operator has the sequence asdepicted in SEQ ID No: 3 or SEQ ID No:
 4. 2. A recombinant adenoviruswherein a gene of viral origin is placed under the control of aninducible promoter, wherein the inducible promoter comprises a minimalpromoter, wherein the minimal promoter has the sequence as depicted inSEQ ID No: 1 or SEQ ID No:
 2. 3. A recombinant adenovirus wherein a geneof adenoviral origin is placed under the control of an induciblepromoter, wherein the gene of adenoviral origin is from the adenovirusE2 region and is represented by a fragment chosen from the groupconsisting of a 72K cDNA, a 140K polymerase cDNA, and a 87K pre-terminalprotein cDNA.
 4. A recombinant adenovirus wherein a gene of adenoviralorigin is placed under the control of an inducible promoter, wherein thegene of adenoviral origin is from the E4 region and is represented by aTaq I-BglII fragment corresponding to nucleotides 35576-32490 ofadenovirus Ad5 genome.
 5. The recombinant adenovirus according to claim4, wherein the gene of adenoviral origin is from the E4 region and isrepresented by the BglII fragment lying-between positions 34115 and32490 of the adenovirus Ad5 genome, containing the sequences of ORF6 andORF7.
 6. A recombinant adenovirus wherein a gene of adenoviral origin isplaced under the control of an inducible promoter, wherein theadenoviral gene is from the region coding for lVa2 and is selected fromthe group consisting of a fragment chosen from a Bgl II-Nru I fragmentcorresponding to nucleotides 3328 to 6316, a Dra I-Nlal II fragmentcorresponding to nucleotides 4029 to 5719, and a Dra I-Xho I fragmentcorresponding to the fragment 4029 to 5788, on the Ad5 adenovirussequence.
 7. A recombinant adenovirus wherein a gene of adenoviralorigin is placed under the control of an inducible promoter, and whereinthe adenoviral gene is a gene of the E2 region, and the induciblepromoter is an Op₂/Tk inducible promoter.
 8. A recombinant adenoviruswherein a gene of viral origin is placed under the control of an Op2Tkinducible promoter, wherein the viral gene is an adeno-associated virus(AAV) gene.
 9. The recombinant adenovirus according to claim 8, whereinthe AAV gene encodes encapsidation functions of an adeno-associatedvirus (AAV).
 10. The recombinant adenovirus according to claim 8,wherein the Op2/Tk inducible promoter has the sequence as depicted inSEQ ID No. 5.